Arenaviruses merit significant interest both as tractable experimental model systems to study acute and persistent viral infections and as clinically important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans, whereas the prototypic Arenavirus LCMV is a superb workhorse for the investigation of basic concepts in the fields of viral immunology and pathogenesis. Moreover, evidence indicates that LCMV is a neglected human pathogen of clinical significance. Our long-term objective is to obtain a detailed understanding of the Arenavirus molecular and cell biology. This knowledge will contribute to the elucidation of Arenavirus-host interactions and associated diseases, and facilitate the development of effective antiviral strategies to combat Arenavirus infections. To this end we have developed a robust reverse genetics system for LCMV that provides us with a novel and powerful approach for the investigation of Arenavirus biology. The focus of this proposal is to functionally characterize viral and cellular proteins, and their interactions, which mediate control of Arenavirus RNA synthesis, particle formation and viral budding. Our specific aims are: 1) To Assess The Role Of Z-NP In Control Of Viral RNA Synthesis. We will test the hypothesis that Z-NP interaction is responsible for the documented inhibitory activity of Z on viral RNA synthesis. We will use biochemical and genetic approaches to identify the regions of Z and NP required for Z-NP interaction, and use reverse genetic approaches to examine the functional consequences of disrupting Z-NP interaction. 2) Determine the role of Z-GP interaction in production of arenavirus infectious particles. We have shown that Z and GP are required for production of infectious VLP, and obtained evidence that Z and GP i nteract. We will define biochemically and functionally this Z-GP interaction. We will test the hypothesis that the correct Z-GPcx association is required for the production of infectious particles, and that this interaction is mediated by defined regions w ithin Z and the GP-2 component of the GP complex (GPcx). 3) Determined the function of Z L domain motifs during the natural course of Arenavirus infection. We will use reverse genetics to rescue rLCMV carrying Z proteins with different types of L-domains. These rLCMV will be used in cell culture and mice models of infection to test the hypothesis that the type of L-domain motifs present in Z influences viral growth and virulence. 4) Identify and functionally characterize host proteins that influence Z-mediated budding. We will test the hypothesis thatZ proteins containing different L-domains engage distinct subsets of Class E proteins of the MVB pathway, and that these differences influence virus-host interactions and associated diseases. To identify additional Z-interacting host cell proteins that influe nee Arenavirus budding, we will use TAP combined with MS procedures to define the Z interactome. We will use biochemical, genetics and functional assays to assess the relevance in Arenavirus biology of candidates initially identified by TAP/MS.